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Generating correlated data for omics simulation.

Simulation of realistic omics data is a key input for benchmarking studies that help users obtain optimal computational pipelines. Omics data involves large numbers of measured features on each sample and these measures are generally correlated with each other. However, simulation too often ignores these correlations, perhaps due to computational and statistical hurdles of doing so. To alleviate this, we describe three approaches for generating omics-scale data with correlated measures which mimic real datasets. These approaches are all based on a Gaussian copula approach with a covariance matrix that decomposes into a diagonal part and a low-rank part. This decomposition allows for extremely efficient simulation, overcoming a hurdle for adoption of past methods. We use these approaches to demonstrate the importance of including correlation in two benchmarking applications. First, we show that variance of results from the popular DESeq2 method increases when dependence is included. Second, we demonstrate that CYCLOPS, a method for inferring circadian time of collection from transcriptomics, improves in performance when given gene-gene dependencies in some circumstances. We provide an R package, dependentsimr, that has efficient implementations of these methods and can generate dependent data with arbitrary marginal distributions, including discrete (binary, ordered categorical, Poisson, negative binomial), continuous (normal), or with an empirical distribution.

Computer Simulation

Investigating the causal role of smoking in gout: A triangulation approach combining NHANES data, genetic correlation, and Mendelian randomization.

The relationship between smoking and the development of gout is not well understood. To address this, we adopted a triangulation framework that integrates observational analysis, genetic correlation estimation, and two-sample Mendelian randomization (MR) to examine whether smoking confers a causal risk for gout. We first performed a cross-sectional analysis using information for 13,626 participants from the National Health and Nutrition Examination Survey between 2013 and 2018. The association of smoking with gout was subsequently assessed through logistic regression models. We next investigated the extent of shared genetic factors between smoking phenotypes and gout. We were able to demonstrate this using the linkage disequilibrium score regression applied to genome-wide association study data of European ancestry. Finally, to verify the causality of our relationship, we carried out a two-sample MR analysis. We selected the inverse-variance weighted (IVW) method and confirmed the consistency of using the IVW method with other statistical methods, including weighted median, weighted mode, and simple mode, as well as MR-Egger regression. We performed sensitivity analyses to investigate the heterogeneity of the hypothesis and stability of the data. Our findings based on National Health and Nutrition Examination Survey data reveal that there is a strong positive association between smoking and the risk of gout (odds ratio [OR]&#x2005;=&#x2005;1.94, 95% confidence interval [CI]&#x2005;=&#x2005;1.48-2.55, P&#x2005;<&#x2005;.001). This association persisted after confounding adjustments (OR&#x2005;=&#x2005;1.41, 95% CI&#x2005;=&#x2005;1.04-1.91, P&#x2005;=&#x2005;.027). In the subgroup analyses, former smokers and current smokers of 10 to 20 cigarettes per day had a substantially increased risk. Post-linkage disequilibrium score regression analysis revealed that the significantly positive genetic correlations of smoking initiation and lifetime smoking index with gout risk were both significantly positive. Additional evidence for causality is presented by MR. Genetic prediction of smoking initiation statistically increases gout risk (IVW OR&#x2005;=&#x2005;1.55, 95% CI&#x2005;=&#x2005;1.26-1.90, P&#x2005;=&#x2005;3.17&#x2005;&#xd7;&#x2005;10-5). A much stronger association is evident for lifetime smoking index (IVW OR&#x2005;=&#x2005;1.99, 95% CI&#x2005;=&#x2005;1.44-2.76, P&#x2005;=&#x2005;3.24&#x2005;&#xd7;&#x2005;10-5). These findings are the same with or without heterogeneity by sensitivity analysis. In light of our integrated analysis, smoking is a causative factor for gout. This suggests that public health interventions like anti-smoking campaigns might reduce gout incidence.

Humans

Probiogenomic analysis of functional potential and safety of L. plantarum 8p-a3 and DMC-S1 strains: in silico vs in vitro and in vivo data.

The molecular basis of the beneficial effects and the causes of the negative effects of probiotics are not entirely clear. Clarifying these issues is important for understanding the biology and assessing the safety of the microbes. Omics technologies have opened up new resources for obtaining relevant knowledge. Here, for the first time, we present the results of a comparative analysis of the functional potential and safety of two L. plantarum strains: the approved probiotic 8p-a3 and the Drosophila intestinal resident, which exhibit opposite effects on D. melanogaster as the model host organism. Through genomic analysis, extracellular vesicle studies, and in vitro and in vivo assays, we have identified the common and specific characteristics of the strains. The strains proved to be similar in a set of genes that determine benefits to the host organism, as well as in the presence of some risk factors. Significant differences between the strains are related to genes responsible for adhesion, sialic acid metabolism, mucin degradation, antimicrobial peptides, tannin resistance, and immunomodulation. In silico data correlated with in vitro and in vivo data, with the exception of antimicrobial sensitivity. Pronounced differences between the strains were found in terms of the composition and biological effects of their vesicles. In vivo data on the effects of the strains correlate with the corresponding data of their vesicles in the fruit fly model. The results obtained open up new facets in L. plantarum strains relevant for evaluating the functionality and safety of probiotics.IMPORTANCEUsing a probiogenomic approach, common and specific features regarding functionality and safety were identified in the strains (the approved probiotic strain L. plantarum 8p-a3 and the Drosophila intestinal bacterium L. plantarum DMC-S1), which exhibit opposite effects on the model host organism (D. melanogaster). The genomic analysis was supplemented by the analysis of extracellular vesicles of the strains. Comparative analysis of in silico data in combination with in vitro and in vivo studies was performed, and unexpected capabilities of the strains were discovered. Novel factors, essential for evaluating the safety of probiotics, were identified. New facets in the interplay of probiotic bacterium with host organism have been revealed.

Animals

Proportionality-based association metrics in count compositional data.

Compositional data comprise vectors that describe the constituent parts of a whole. Data arising from various -omics platforms such as 16S and RNA sequencing are compositional in nature. In this kind of data, correlations between features on raw counts have no meaningful interpretation. Metrics of proportionality were formulated to address this problem. However, an inherent bias arises when these metrics are calculated empirically on count-based measures due to variability in read depths. We quantify the bias introduced by empirically calculating proportionality-based association metrics in count data. Additionally, we propose a means of estimating these metrics within a logit-normal multinomial model in pursuit of more accurate estimates. The model-based estimates are shown to outperform empirical estimates in simulated data and are applied to a mouse embryonic stem cell single-cell sequencing dataset, as well as a pediatric-onset multiple sclerosis metagenomic dataset.

Animals

Cardiomyocyte-Specific Plakophilin-2 Loss Is Sufficient to Induce Aging and Senescence of Nonmyocytes: Relevance to Arrhythmogenic Cardiomyopathy.

BACKGROUND: Pathogenic variants in PKP2 are the most common cause of familial arrhythmogenic right ventricular cardiomyopathy. This study tests whether plakophilin-2 (PKP2) deficiency only in cardiomyocytes is sufficient to provoke premature aging and proinflammatory senescence in nonmyocyte, cardiac resident cells. METHODS: We studied mice with cardiomyocyte-specific, tamoxifen-activated loss of PKP2 (cardiomyocyte-specific conditional knockout of plakophilin-2) using conventional and multiplex imaging, cytokine arrays, epigenetic clocks, spatial transcriptomics, expansion and structured illumination microscopy, and correlative data analysis. We examined nonmyocytes and cardiomyocytes for premature aging and senescence. RESULTS: We observed senescence-associated heterochromatin foci in nonmyocytes, predominantly in cells positive for &#x3b1;-smooth muscle actin staining. Cytokines in media of nonmyocyte cells were consistent with senescence-associated secretory phenotype. Epigenetic clocks identified premature aging. Multiplex immunohistochemistry showed nonmyocyte cells in niches, intermingled with cardiomyocytes. Spatial transcriptomics showed overrepresentation of senescence-associated secretory phenotype-related transcripts, predominantly in myocyte-rich areas of the left ventricle. Senescence-associated heterochromatin foci and increased epigenetic age were not found in cardiomyocytes from cardiomyocyte-specific conditional knockout of plakophilin-2 hearts, although we observed structural features associated with premature aging. Cross-reference analysis showed correlation between the cardiomyocyte-specific conditional knockout of plakophilin-2 cardiac proteome and that of mice 5 or 6 times their chronological age, as well as transcriptional signatures of neurodegenerative diseases. CONCLUSIONS: Loss of PKP2 expression only in adult cardiac myocytes is sufficient to induce proinflammatory senescence in nonmyocytes, and overall premature cardiac aging. This is the first study to intersect cellular senescence and premature aging with desmosomal arrhythmogenic cardiomyopathies. We speculate that cell-agnostic molecular signatures, biomarkers, and pharmacology of senescence and of neurodegenerative diseases may be relevant to diagnose or treat PKP2 arrhythmogenic right ventricular cardiomyopathy.

Animals

Methylation-based droplet digital polymerase chain reaction shows high concordance with chronic lymphocytic leukemia IGHV somatic mutation status.

OBJECTIVE: Somatic hypermutation at immunoglobulin heavy chain variable (IGHV) genes, an established prognostic and predictive biomarker for chronic lymphocytic leukemia (CLL), is assessed by gene sequencing. We developed a single methylation-specific droplet digital polymerase chain reaction (methyl-ddPCR) to predict IGHV status in patients with CLL. METHODS: The CLL methylation array and IGHV data from the International Cancer Genome Consortium (ICGC) were used for biomarker discovery. Top-ranked candidate regions were manually screened for PCR primer and probe binding sites. A single methyl-ddPCR was evaluated on an internal cohort of CLLs with mutated (M), unmutated (U), and inconclusive IGHV results originally determined by next-generation sequencing (NGS). RESULTS: Analysis of ICGC data identified array probe cg23844018 as a candidate for the PCR. The corresponding CpG site showed high methylation levels in U-CLL and lower levels in M-CLL. On the internal cohort, a single optimal cutoff correctly classified 104 of 115 U- and M-CLLs (90.4%; area under the curve&#x2005;=&#x2005;0.96). The PCR data correlated with some prognostic fluorescence in situ hybridization and CLL subset groupings. Limited analysis suggests that the PCR may be able to stratify some patients with CLL who have inconclusive results on IGHV NGS testing. CONCLUSIONS: The methyl-ddPCR showed high concordance with CLL IGHV status in an internal cohort.

Humans

Systematic functional evaluation of CNGA1 missense variants associated with retinitis pigmentosa.

BACKGROUND: Missense variants are frequently classified as variants of uncertain significance (VUS) according to the guidelines of the American College of Medical Genetics and Genomics and the Association of Molecular Pathology (ACMG/AMP). Consequently, disease relevance remains elusive, impeding molecular genetic diagnostics, patients` and family genetic counseling, and identification of patients eligible for clinical trials. Functional studies are critical for resolving the clinical significance of VUS. CNGA1 encodes the main subunit of the rod cyclic nucleotide-gated (CNG) channel, a vital component of the phototransduction cascade. Variants in CNGA1 are a rare cause of autosomal recessive retinitis pigmentosa and a phase I/II gene augmentation trial (NCT06291935) is currently ongoing highlighting the necessity to differentiate benign from pathogenic variants. METHODS: CNGA1 missense variants compiled from retinal disease patient cohorts, public databases and literature were functionally investigated using a medium-throughput aequorin-based assay and in vitro minigene splice assays for predicted exonic spliceogenic variants. Functional data were correlated with the in silico prediction of five variant effect predictors (VEPs) and applied to support or revise variants' ACMG/AMP classification. RESULTS: Data mining revealed 86 missense CNGA1 variants - including three novel - most of them lacking functional data; 65.1% of the variants were initially classified as VUS. The aequorin-based assay showed that 72.1% of tested variants significantly impaired CNG channel function and were classified as functionally abnormal, while 23.3% were functionally normal and 5% remained functionally uncertain. Correlation of the functional data with in silico predictions identified AlphaMissense and CPT-1 to be the most suitable tools for assessing CNGA1 missense variants. Using in vitro minigene splice assays, two putative missense variants were shown to induce missplicing. Based on the functional findings, 62.1% of the variants initially classified as VUS were re-categorized as likely pathogenic or likely benign. Furthermore, 93.3% of the variants initially classified as likely pathogenic showed an effect on CNGA1 channel function, confirming their disease relevance and supporting their reclassification as pathogenic. CONCLUSION: This study represents the first comprehensive functional assessment of disease-associated CNGA1 missense variants, thus significantly advancing the understanding of their disease relevance and improving molecular genetic diagnostics in patients.

Humans

RPN1 at the crossroads of glycosylation, tumor immunity, and disulfidptosis.

Ribophorin I (RPN1), a core component of the oligosaccharyltransferase complex, is traditionally known for its role in endoplasmic reticulum-associated N-glycosylation. Recent studies have identified RPN1 as an emerging regulator of tumor progression and immunity. Aberrant RPN1 overexpression has been reported in multiple malignancies, including glioma, hepatocellular carcinoma, sarcoma, and triple-negative breast cancer, where it is frequently associated with aggressive clinicopathological features and poor prognosis. RPN1 promotes tumor immune evasion by promoting N-glycosylation and stabilization of programmed death-ligand 1 (PD-L1), thereby enhancing immune checkpoint signaling and directly inhibiting anti-tumor T-cell responses. Consequently, elevated RPN1 expression is consistently associated with an immunosuppressive tumor microenvironment rich in M2 macrophages and poor in CD8+ T cells. More importantly, multiple omics signature analyses indicate RPN1 is integrated into several disulfidptosis-related risk models; however, direct experimental evidence confirming the causal linkage between RPN1 and disulfidptosis remains limited. Correlative database data also show potential associations between RPN1 upregulation and genomic instability and treatment resistance. Based on tiered classification of existing evidence (biochemical functional validation vs. multi-omics correlation), this review systematically summarizes the biological roles of RPN1 in cancer, its functions in tumor immunity and disulfidptosis-associated pathways and finally evaluates its potential as a therapeutic target in precision oncology.

PDL1

Quantitative natural history modeling of HPDL-related disease based on cross-sectional data reveals genotype-phenotype correlations.

PURPOSE: Biallelic HPDL variants have been identified as the cause of a progressive childhood-onset movement disorder, with a broad clinical spectrum from severe neurodevelopmental disorder to juvenile-onset pure hereditary spastic paraplegia type 83. This study aims at delineating the geno- and phenotypic spectra of patients with HPDL-related disease, quantitatively modeling the natural history, and uncovering genotype-phenotype associations. METHODS: A cross-sectional analysis of 90 published and 1 novel case was performed, using a Human-Phenotype-Ontology-based approach. Unsupervised phenotypic clustering was used alongside in silico analyses to identify distinct patient subgroups. RESULTS: The study models the natural history of the HPDL-related disease in a global cohort, clarifying the molecular and phenotypic spectrum and identifying 3 distinct subgroups characterized by differences in onset, clinical trajectories, and survival. It establishes genotype-phenotype associations, showing that the presence of moderately pathogenic missense variants in 1 allele leads to a milder, spastic paraplegic phenotype with later disease onset, whereas biallelic, highly pathogenic missense or truncating variants are associated with a more severe phenotype and reduced life span. CONCLUSION: Quantitative and unbiased natural history modeling in HPDL-related disease reveals significant genotype-phenotype associations, providing a foundation for variant interpretation, anticipatory guidance, and choice of outcome measures in future prospective and functional studies.

Humans

Neuroimaging PheWAS and molecular phenotyping implicate PSMC3 in Alzheimer's Disease.

INTRODUCTION: Neuroimaging genetics have advanced Alzheimer's disease (AD) research, yet frameworks mechanistically connecting genes to neurological outcomes via functional genomics are needed to elucidate genetic associations. To address this challenge, we assessed relationships between AD-associated variants and disease via their impact on gene expression and neuroimaging phenotypes. METHODS: We mapped established AD genes to neuroimaging traits using NeuroimaGene atlas and predicted transcript-driven AD neurological features by comparing gene-derived neuroimaging features to clinical neuroimaging data. Genetic correlation and covariance analyses characterized shared genetic architecture between AD endophenotypes and neuroimaging features and identified neuroimaging features associated with dementia family history. RESULTS: Our analyses implicate PSMC3 expression as a strong contributor to AD pathophysiology and indicate AD endophenotypes, including dementia family history, linked to frontal cortex thickness, volume, and cerebrospinal fluid volume changes. DISCUSSION: Our findings prioritize AD genes whose regulation is associated with vulnerable brain regions, offering a potential mechanistic framework for downstream functional validation.

Alzheimer&#x2019;s Disease

Natural leaf shape variation reveals diverse transcriptional targets of GmJAG1 during soybean leaf development.

The JAGGED transcription factor family regulates lateral organ development across angiosperms. In soybean (Glycine max Merr.), a D9H mutation in the EAR repression motif of GmJAG1 causes a narrow-leaflet phenotype and explains over 70% of phenotypic variance in leaf shape. Because this mutation does not affect the zinc finger DNA-binding domain, both alleles bind identical targets but differ in repressor recruitment. Previous studies mapped GmJAG1 binding sites, but the functional targets controlling leaf morphology are uncharacterized. Here, we used comparative transcriptomics across four soybean genotypes with contrasting leaf shape, spanning a developmental time series from shoot apex to mature leaf, and identified 1567 putative candidate target genes. GmJAG1 expression was confined to the shoot apex, yet 99.1% of candidate targets maintained differential expression throughout development. We found that neither Kip-Related Protein (KRP) cell cycle inhibitors nor Cyclin-Dependent Kinases (CDKs) showed differential expression despite binding evidence in Arabidopsis. However, D-type cyclins were upregulated in narrow-leaf genotypes suggesting that soybean GmJAG1 acts through cyclin-mediated rather than KRP-mediated cell cycle regulation described in Arabidopsis- a divergence in regulatory logic between the two species. Pathway analysis revealed enrichment of auxin (1.8-fold, P&#x2009;=&#x2009;0.02) and salicylic acid (fourfold, P&#x2009;=&#x2009;0.016) genes among JAG1D9H targets. Filtering by differential expression, binding data, phenotype correlation, and co-expression network membership identified 79 high-confidence targets, including orthologs of NPH3 (phototropin-mediated leaf flattening), MIK2 (cell wall integrity sensing), RD22 (ABA-responsive stress signaling), and SCL23 (GRAS transcription factor in bundle sheath development). These candidates provide targets for functional validation and breeding in legumes.

Glycine max

Estimating population structure using epigenome-wide methylation data.

INTRODUCTION: In epigenome-wide association analysis (EWAS), unaddressed population stratification often leads to inflation. We aimed to compute methylation population scores (MPSs) that predict genetic principal components (GPCs) using a feature selection and regression approach. METHODS: We used multi-ethnic methylation data (Illumina 450K/EPIC array) from unrelated MESA (n=929), CARDIA (n=1123), JHS (n=1365), ARIC (n=2338), and HCHS/SOL (n=1475) individuals, randomly assigning 85% of participants from each cohort to a training dataset and the remaining 15% to a test dataset. First, we estimated the associations of GPCs with each available CpG methylation site using linear regression within each cohort, adjusting for age, sex, smoking status, race/ethnic background (as a proxy for background information associated with lifestyle and other environmental exposures that may impact methylation), alcohol use status, body mass index, and cell type proportions. We meta-analyzed the associations across cohorts and selected CpG sites with association FDR-adjusted q-value <0.05. We next aggregated individuallevel data across the cohort-specific training datasets, and applied two-stage weighted least squares Lasso regression, with the GPCs as the outcomes and the selected CpG sites as penalized predictors, adjusting for the aforementioned covariates. The developed MPSs are the weighted sum of selected CpG sites from the Lasso. To evaluate the developed MPSs, we constructed them in the test dataset, and compared them with GPCs, and with MPSs constructed based on a previously-published paper. Comparison was based on correlation analysis and data visualization. We demonstrate the use of the MPSs in EWAS. RESULTS: In the test dataset, the MPSs were highly correlated with GPCs, with correlation decreasing, though not monotonically, for later components. Specifically, MPS1 and GPC1 had R2= 0.99, while MPS7 and GPC7 had R2=0.27 (the lowest observed correlation). In data visualization, MPSs had similar patterns as GPCs in differentiating self-reported White, Black, and Hispanic/Latino groups, while outperforming MPC constructed using alternative published methods. MPSs showed comparable performance to GPCs in reducing some of the inflation in EWAS. CONCLUSIONS: Methylation-based population scores provide a reliable estimate of population structure in the data and can complement GPCs when genetic data are absent. Unlike previous methods based on unsupervised methylation PCA, MPSs uses supervised learning with covariate adjustment to capture genetic structure across diverse populations. The weights for each GPCs derived in our study can be applied to generate MPSs in other studies.

Journal Article

Survival prediction for clear cell renal cell carcinoma based on deep multimodal synergistic survival network.

Objective.To propose a deep multimodal synergistic survival analysis framework (Deep Multimodal Synergistic Survival Network, DMSSN) to achieve accurate prognostic analysis for clear cell renal cell carcinoma (ccRCC).Methods.This study (DMSSN) utilized matched multimodal data from the Cancer Genome Atlas-KIRC database, including CT imaging data, whole slide images, copy number variation (CNV) features, and clinical data. Deep Canonical Correlation Analysis was employed to map heterogeneous modalities into a shared latent space. Contrastive learning was introduced to enhance semantic consistency across multimodal features, and a gating network was utilized for the adaptive fusion of multimodal information to achieve precise survival risk prediction for patients.Results.Experimental results demonstrated that DMSSN achieved a Concordance Index (C-index) of 0.8153 &#xb1; 0.0994, with a Log-rank testp-value of 1.6553&#xd7;10-11. DMSSN exhibited significant performance advantages over traditional statistical methods like Log-rank-Cox (0.7055 &#xb1; 0.0670) and machine learning methods such as Random Survival Forest (RSF) (0.6836 &#xb1; 0.1048). Furthermore, in comparison with similar deep learning approaches, DMSSN outperformed late fusion strategies (0.7493 &#xb1; 0.1211) and discrete-time survival models such as DeepHit (0.7655 &#xb1; 0.1041) and Nnet-surv (0.7694 &#xb1; 0.0635). Notably, DMSSN still achieved the best predictive performance when compared to the classic deep survival model DeepSurv (0.7919 &#xb1; 0.0978) and advanced state-of-the-art multimodal fusion frameworks like Context-Aware Transformer (0.7735 &#xb1; 0.0818) and Multimodal Co-Attention Transformer (0.8102 &#xb1; 0.0972). Ablation studies showed that removing any single modality led to a decline in performance, with the largest numerical decrease occurring after removing CT imaging features (C-index decreased to 0.7327), validating the complementarity of multimodal data and the pivotal role of radiomic features in prognostic assessment. Module ablation experiments further confirmed the effectiveness of the core components.Conclusion:By effectively integrating imaging, pathology, genomic, and clinical features, the DMSSN framework demonstrates superior performance and robustness in the survival prediction of ccRCC.

Carcinoma, Renal Cell

Application of SPI-guided analgesia in laparoscopic gynecologic surgery: a randomized controlled trial evaluating the remifentanil-sparing effect and predictive value of time-weighted SPI.

This study aimed to achieve two primary objectives: (1) to evaluate the opioid-sparing effect of Surgical Pleth Index (SPI)-directed analgesia during surgery via a randomized controlled trial (RCT), and (2) to propose and preliminarily assess a novel dynamic metric, Threshold-based Time-Weighted SPI (Tb-TW-SPI), which integrates stimulus intensity and duration, for its predictive efficacy regarding postoperative moderate-to-severe pain. Employing an RCT combined with exploratory analysis, 61 patients undergoing elective laparoscopic gynecologic surgery were randomized into an SPI-directed analgesia group or a conventional analgesia group. The primary outcome was total intraoperative remifentanil consumption. Postoperatively, an exploratory analysis of the control group data evaluated the correlation between Tb-TW-SPI and Numeric Rating Scale (NRS) pain scores in the post-anesthesia care unit (PACU), calculating its predictive value for moderate-to-severe pain (NRS&#x2009;&#x2265;&#x2009;4). Results: The SPI-directed group required significantly less intraoperative remifentanil than the conventional group [median (IQR): 5.84(5.02,6.62)vs. 6.96(5.81,8.19)&#xb5;g/kg/h; P&#x2009;=&#x2009;0.016]. Postoperative pain scores did not differ significantly between groups (P&#x2009;>&#x2009;0.05). Exploratory analysis of the conventional analgesia group revealed that Tb-TW-SPI values were significantly higher in patients with moderate-to-severe postoperative pain (NRS&#x2009;&#x2265;&#x2009;4) compared to those without (P&#x2009;=&#x2009;0.0417).The area under the ROC curve for Tb-TW-SPI predicting this pain was 0.74 (95% CI: 0.52-0.96), with 67% sensitivity and 76% specificity at an optimal cutoff of 1210. This RCT suggests that SPI-directed analgesia can safely and moderately reduce intraoperative remifentanil consumption. Furthermore, the proposed Tb-TW-SPI metric, in this exploratory analysis, suggests potential for predicting postoperative pain, though this finding requires validation in larger cohorts with higher-frequency SPI sampling, offering a new direction for SPI interpretation. Large-scale, multicenter trials are warranted to validate the predictive utility of Tb-TW-SPI. Clinical Trial Registration, China Clinical Trial Registry: ChiCTR2400088444.

Humans

Novel splice site variants in GBA1 are associated with Gaucher disease and genotype-phenotype correlations.

BACKGROUND: Variants in GBA1 are associated with neurodegenerative disease. This study aimed to explore pathogenic GBA1 variants. METHODS: Four patients with progressive myoclonic epilepsy (PME) and extremely low &#x3b2;-glucosidase levels were recruited. Whole-exome sequencing and long-range PCR were performed to identify GBA1 variants. Bioinformatic analyses were used to predict the impact of the identified variants. A literature review was performed to explore the genotype-phenotype correlations. GBA1 expression data across different brain regions and developmental stages were analyzed using the BrainSpan database. RT-PCR was performed to verify the splicing effects. RESULTS: Compound heterozygous GBA1 variants were identified in four patients. Five distinct variants were detected, including two novel splice site variants (c.308-2A>G and c.762-2A>C) and three previously reported variants. All identified variants were rare or absent in gnomAD. Splice site variants c.308-2A>G and c.762-2A>C were predicted to cause aberrant splicing. Minigene-based splicing assays coupled with RT-PCR and Sanger sequencing confirmed that both variants cause complete exon skipping (exon 4 and exon 7, respectively). All patients presented with PME onset in childhood/adolescence, intellectual regression, low &#x3b2;-glucosidase, and diffuse brain atrophy and were subsequently diagnosed with Gaucher disease type 3. GBA1 expression in the brain showed two distinct peaks: one in infancy and another after five years of age. The onset age of PME aligned with the second GBA1 expression peak (after five years of age). CONCLUSION: This study identified compound heterozygous GBA1 variants, including two novel candidate pathogenic splice site variants, in Gaucher disease type 3 patients, expanding the known mutational spectrum.

Humans

abCRISPR: deep learning-based design of abasic gRNA sequences for specific CRISPR-Cas9 genome editing.

SUMMARY: CRISPR-Cas9 has become a widely used tool for genome editing. However, its off-target cleavage caused by partial sequence matches with guide RNAs (gRNAs) remains a critical limitation. Recently, abasic gRNAs (&#xd8;X&#xd8;) have been developed to enhance target specificity, but their effects vary depending on the positional sequence context. Here, we present abCRISPR, a deep neural network (DNN) framework for the rational design of &#xd8;X&#xd8; sequences with minimized off-target activity. abCRISPR leverages informative few-shot training with paired datasets of abasic and unmodified gRNAs, using high-quality random mismatch target libraries, exhaustively sequenced for mismatched off-target substrates (n&#x2009;=&#x2009;97583) in in vitro CRISPR-Cas9 cleavage experiments. Predicted off-target activities for both abasic and unmodified gRNAs showed strong correlation with experimental data (r&#x2009;&#x2265;&#x2009;0.95, 10-fold cross-validation). Notably, these comprehensive training sets provide robust ground-truth negatives, enabling accurate and sensitive prediction of off-targets. For unmodified gRNAs, abCRISPR (AUC = 0.98) was validated to outperform existing deep learning-based methods (AUC = 0.45-0.68). When applied to the human genome, abCRISPR generated &#xd8;X&#xd8; sequences, covering 58&#xa0;875&#xa0;004 potent CRISPR-targetable sites with improved target specificity. Together, this work provides a comprehensive bioinformatics resource for safe and precise CRISPR-Cas9 genome editing. AVAILABILITY AND IMPLEMENTATION: The source code for abCRISPR and training data are available at https://doi.org/10.5281/zenodo.20398246. abCRISPR results for the human genome are available at http://clip.korea.ac.kr/abCRISPR/.

Deep Learning

The role of estrogen receptors and house dust mite-induced DNA methylation in a mouse model.

Asthma is a chronic respiratory disease affecting over 230 million people worldwide, with higher prevalence in women. Environmental allergens such as house dust mite (HDM) trigger airway inflammation and hyperresponsiveness (AHR), yet the epigenetic mechanisms underlying these responses remain poorly understood. Furthermore, the role of estrogen receptors in the context of asthma is understudied. We aimed to investigate whether estrogen receptor-specific DNA methylation contributes to HDM-induced airway remodeling and hyperresponsiveness. Male and female C57BL/6J wild-type mice and estrogen receptor &#x3b1; and &#x3b2; knockout mice (Esr1-/- and Esr2-/-) were exposed to HDM or phosphate-buffered saline for 5 wk. DNA methylation and RNA sequencing data were obtained from snap-frozen whole lung tissues. HDM exposure resulted in widespread differential methylation of genes associated with inflammation and AHR, including Itgal, Tmem267, Rap1b, Bmf, Mid1, Fgd1, Ddx4, Comtd1, Filip1l, Grb10, and Chst7. Notably, the absence of estrogen receptor &#x3b2; (in Esr2-/- mice) produced the most pronounced methylation patterns, particularly in females. Pathway enrichment analysis revealed asthma-relevant processes such as extracellular matrix remodeling, leukocyte adhesion and migration, airway smooth muscle contraction, and inflammatory signaling. Integration of methylation and gene expression data confirmed significant correlations (P < 0.05) for Itgal, Rap1b, and Tmem267, and a marginal correlation for Chst7 (P < 0.1), implicating these genes in allergic asthma pathogenesis. Our findings demonstrate that HDM exposure induces sex-specific epigenetic changes mediated by estrogen receptor status, highlighting a potential mechanism for increased asthma susceptibility in women. These results can inform estrogen receptor-targeted treatment strategies for allergic airway diseases.NEW & NOTEWORTHY Understanding estrogen receptor-mediated epigenetic regulation provides a foundation for developing sex-specific interventions for asthma, addressing the higher prevalence and severity observed in women. In this study, we demonstrate that exposure to house dust mite in the mouse lung is associated with epigenetic alterations in genes linked to airway hyperresponsiveness and lung inflammation. These alterations were dependent on the presence or absence of estrogen receptors.

Animals